Biochemistry 1980,19, 5339-5344 D3 at high substrate concentrations (Bjorkhem & Holmberg, 1978). Because the microsomes are believed to be the major physiological site of 25-hydroxylation (Madhok & DeLuca, 1979), inhibition of the in vitro microsomal system would lend support for the latter mechanism, Inhibition was observed with a 100-fold excess of inhibitor (Figure 2) in such an assay. The unique inhibitory properties of 19-OH-1O(S), 19-DHD3 therefore may result from its selective inhibition of a microsomal vitamin D3-25-hydroxylase in conjunction with unaffected mitochondrial or exohepatic 25-hydroxylases. However, this will require a more direct investigation before it can be accepted. Acknowledgments We thank M. A. Fivizzani, L. E. Reeve, and C. M. Smith for their excellent technical assistance in performing the bioassays. References Bhattacharyya, M. H., & DeLuca, H. F. (1974a) Biochem. Biophys. Res. Commun. 59, 734. Bhattacharyya, M. H., & DeLuca, H. F. (1974b) Arch. Biochem. Biophys. 160, 58. Bjorkhem, I., & Holmberg, I. (1978) J . Biol. Chem. 253, 842. Bjorkhem, I., Hansson, R., Holmberg, I., & Wikvall, K. (1 979) Biochem. Biophys. Res. Commun. 90, 6 15. Bligh, E. G., & Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911.
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Bradford, M. M. (1976) Anal. Biochem. 72, 248. DeLuca, H. F., Paaren, H. E., & Schnoes, H. K. (1979) Top. Curr. Chem. 83, 1-65. Holick, M. F., Kasten-Schraufrogel,P., Tavela, T., & DeLuca, H. F. (1975) Arch. Biochem. Biophys. 166, 63. Holick, S . A., Holick, M. F., Tavela, T. E., Schnoes, H. K., & DeLuca, H. F. (1976) J . Biol. Chem. 251, 1025. Lund, J., & DeLuca, H. F. (1966) J . Lipid Res. 7 , 739. Madhok, T. C., & DeLuca, H. F. (1979) Biochem. J . 184, 491. Martin, D. L., & DeLuca, H. F. (1969) Am. J . Physiol. 216, 1351. Mourino, A., & Okamura, W. H. (1978) J . Org. Chem. 43, 1653. Norman, A. W., Hammond, M. L., & Okamura, W. H . (1977) Fed. Proc., Fed. A m . SOC.Exp. Biol. 36, 914. Onisko, B. L., Schnoes, H. K., & DeLuca, H. F. (1979a) J . Biol. Chem. 254, 3493. Onisko, B. L., Schnoes, H. K., DeLuca, H. F., & Glover, R. S. (1979b) Biochem. J . 182, 1. Paaren, H. E. (1976) Ph.D. Thesis, University of Illinois, Chicago Circle. Ponchon, G., & DeLuca, H. F. (1969) J . Clin. Inuest. 48, 1273. Sulimovici, S., Roginsky, M. S . , Duffy, J. L., & Pfeifer, R. F. (1979) Arch. Biochem. Biophys. 195, 45. Tucker, G., 111, Grognon, R. E., & Haussler, M. R. (1973) Arch. Biochem. Biophys. 155, 47.
Changes in Membrane Potential and Membrane Fluidity in Tetrahymena pyriformis in Association with Chemoreception of Hydrophobic Stimuli: Fluorescence Studied Hiroshi Tanabe,* Kenzo Kurihara, and Yonosuke Kobatake
ABSTRACT:
The fluorescence intensity of rhodamine 6G (Rh6G) and 1,6-diphenyl-1,3,5-hexatriene(DPH) in the presence of Tetrahymena pyriformis was measured to monitor changes in the membrane potential and in the gross structure of the surface membrane in response to chemical stimuli. So-called “odorants” for higher vertebrates, which are usually uncharged and hydrophobic compounds, were chosen as chemical stimuli for a model study of the olfactory response. The fluorescence intensity of Rh6G started to increase at the chemotactic thresholds of the stimuli, indicating that negative chemotaxis of T . pyriformis to the hydrophobic stimuli is induced by depolarization of the cell. The fluorescence in-
tensity of DPH increased in close association with chemoreception of the hydrophobic stimuli. The increase in the fluorescence intensity was ascribed mainly to uptake of DPH, suggesting that gross structural changes of the surface membrane occur with the reception of hydrophobic stimuli. The membrane fluidity determined by fluorescence polarization of DPH increased in close association with the chemoreception of the hydrophobic stimuli. Inorganic salts such as NaCl, KCI, and CaC12 did not change the DPH fluorescence intensity or the fluorescence polarization, although these stimuli induced depolarization and negative chemotaxis in T . pyriformis.
L i v i n g organisms from unicellular organisms to higher vertebrates have an ability to recognize chemical stimuli in external environments. The response of unicellular organisms to chemical stimuli can be seen in chemotaxis. In higher vertebrates, chemical stimuli in the external environment are received at gustatory and olfactory cells. Recently much
attention has been paid to the molecular mechanism of chemoreception in the sensory cells, but a detailed mechanism is still unknown. The difficulties encountered in exploring the mechanism come from the limitations of the techniques that can be applied, few techniques besides electrophysiologicalones can be applied to intact sensory cells. Furthermore, in the case of olfactory cells, which are terminal swellings of olfactory nerves, intracellular recordings of electrical properties of the cells are extremely difficult because of the small size of the
From the Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan. Received November 8, 1979.
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0 1980 American Chemical Society
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cells. It is still unknown how “odorants”, which are usually uncharged and hydrophobic compounds, induce changes in the membrane potential of the olfactory cells, Tetrahymena pyriformis is a ciliated protozoan and is considered to have excitable membranes on the basis of the fact that the related organism Paramecium has excitable membranes (Naitoh & Eckert, 1969; Eckert, 1972). T . pyriformis exhibits negative chemotaxis to various inorganic salts and hydrophobic compounds (Tanabe et al., 1979). In a previous paper, we showed that chemoreception of T . pyriformis to so-called odorants for higher vertebrates has a close correlation to the olfactory response in humans (Ueda & Kobatake, 1977). T. pyriformis is a useful model for the chemosensory cells in studying the receptor mechanism at the membrane level, because a large amount of cell suspension is easily available. In recent years, fluorescence probes have been extensively used to investigate the dynamic properties of biological membranes. Fluorescence probes can be classified as “potential probes” or “structural probes”. In a previous paper, we showed that fluorescence changes of rhodamine 6G (Rh6G) well monitor changes in the membrane potential of T . pyriformis in response to inorganic salts (Aiuchi et al., 1980). This method is applied in the present study to monitor changes in the membrane potential in response to certain hydrophobic compounds (odorants for higher vertebrates). We also use 1,6-diphenyl-l,3,5-hexatriene (DPH) to monitor gross structural changes of the surface membrane. It is shown that changes in the membrane potential and the membrane fluidity occur in close association with the reception of hydrophobic compounds. The significance of the membrane fluidity changes in the reception of hydrophobic compounds is discussed. Experimental Procedures Cell Growth. T . pyriformis (strain w) was grown at 22 “ C in the medium containing 2% proteose peptone, 1% yeast extract, and 0.6% glucose. The cells taken from 2- or 3-day-old culture were collected by gentle filtration through a filter paper (Whatman No. 3) and washed thoroughly with 1 mM 2amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride (Tris-HC1) buffer of pH 7.0 (control solution). Isolation of Membrane Fraction. The surface membrane fraction (pellicle) was isolated according to the method of Nozawa & Thompson (1971). The fractionated membranes were resuspended in the control solution and used for the experiments. Fluorescence Measurements. All the fluorescence measurements were made at 22 “C. Fluorescence intensity and polarization were measured with a fluorescence spectrophotometer, Hitachi MPF-2A, equipped with a circulating water bath (Haake). ( I ) Measurements of Rh6G Fluorescence. Rhodamine 6G (Rh6G) dissolved in distilled water was added to a final concentration of 0.7 pM into the cell suspension (1.2 X IO4 cells/mL) and equilibrated at 22 “ C for 20 min. Into a cuvette, 3 mL of the suspension was pipetted for the fluorescence measurement. A given volume of stimulating solution of varying concentrations was added into the cuvette, and the fluorescence intensity was measured. The excitation wavelength was 520 nm and the fluorescence was detected at 550 nm. Some stimulating chemicals were dissolved in ethanol. The final concentration of ethanol never exceeded 1% in any experiment. The fluorescence intensity was not affected by 1% ethanol itself. The change in the fluorescence intensity was defined as eq 1 where F and Fo represent the fluorescence
TANABE, KURIHARA, A N D KOBATAKE
AF = (F - Fo)/F,
(1)
intensities in the presence and absence of stimulating chemicals. ( 2 ) Fluorescence Labeling with DPH. The labeling procedure of T . pyriformis with DPH was carried out according to Shinitzky & Inbar (1974). Two millimolar 1,6-diphenyl1,3,5-hexatriene (DPH) in tetrahydrofuran (THF) was diluted 1000-fold with the control solution under vigorous stirring. Stirring was continued for 20 min. One volume of cell suspension (1.5 X lo4cells/mL) in the control solution was mixed with 1 volume of the DPH dispersion and equilibrated at 22 “ C for 1 h. T. pyriformis in the suspension thus prepared exhibited normal motility for at least 1 day. This indicates that 1 pM DPH and 0.1% T H F did not affect their motility. The suspension of unlabeled cells in the same density was used as a reference sample. The surface membranes (pellicle) were labeled in a manner similar to the case of the intact cells. The equilibration period was 30 min. ( 3 ) Measurements of DPH Fluorescence. Two milliliters of the cell suspension was pipetted into a cuvette and stirred gently with a magnetic stirrer. After 10 min of stirring, hydrophobic compounds dissolved in ethanol or inorganic salts dissolved in water were added to the cuvette. The final concentration of ethanol was
